Compositions and methods for genome-wide mapping of chromosome breakage and other methods for manipulation of cells embedded in matrix

Abstract

The embodiments described herein provide for compositions and methods for genome-wide mapping of chromosome fragile sites in cellular chromosomal material. More specifically, a method of genome-wide detection of regions of single-stranded DNA and double stranded breaks in DNA, the hallmarks of chromosome fragility, comprises embedding a plurality of cells in a matrix and subsequently directly labeling the single-stranded DNA and/or double stranded DNA breaks; eluting or isolating the labeled chromosomal material from the matrix; and performing analysis to detect the location of the chromosome fragility sites.

1 . A method for genome-wide detection of chromosome fragile sites characterized by regions of single-stranded DNA (ssDNA) or double stranded breaks (DSB) in eukaryotic cellular chromosomal DNA, the method comprising:
 (a) obtaining a matrix in which a plurality of cells have been embedded and said cells' chromosomes have been made accessible to labeling reagents;   (b) within the matrix, contacting the chromosomal DNA of the cells with labeling reagents to label directly the DNA at ssDNA regions or at sites of DSB;   (c) isolating the labeled DNA from the matrix; and   (d) detecting the labeled DNA, wherein the detection of labeled DNA permits identification of the location of the ssDNA region or DSB sites and/or the magnitude of labeling at such sites.   
     
     
         2 . The method of  claim 1 , further comprising the step, after labeling step (b), of fragmenting the chromosomal DNA. 
     
     
         3 . The method of  claim 2 , wherein the fragmenting step is performed before or after isolating step (c) 
     
     
         4 . The method of  claim 2 , wherein the fragmenting step comprises mechanical shearing, enzymatic cleavage, or chemical cleavage. 
     
     
         5 . The method of  claim 4 , wherein the fragmenting step produces fragments having an average size of about 400 bp to about 600 bp, inclusive 
     
     
         6 . The method of  claim 1 , wherein the step of isolating labeled DNA from the matrix comprises electroelution or digestion of the matrix. 
     
     
         7 . The method of  claim 1 , wherein detecting step (d) comprises contacting the labeled DNA with a microarray, or analyzing labeled DNA by high throughput DNA sequencing. 
     
     
         8 . The method of  claim 7 , wherein the detecting step permits detection of both the location of the chromosome fragile site and the magnitude of labeling at the chromosome fragile site. 
     
     
         9 . The method of  claim 1  wherein the labeling reagents comprise random primers that permit labeling at ssDNA chromosomal regions. 
     
     
         10 . The method of  claim 1  wherein the labeling reagents comprise Klenow fragment or T4 polymerase 
     
     
         11 . The method of  claim 1  wherein the labeling reagents comprise at least one fluorescently labeled nucleotide. 
     
     
         12 . The method of  claim 11 , wherein the fluorescently labeled nucleotide comprises fluorescently labeled dUTP. 
     
     
         13 . The method of  claim 1 , wherein the cell is a yeast cell or a mammalian cell. 
     
     
         14 . The method of  claim 13 , wherein the mammalian cell is a human cell. 
     
     
         15 . The method of  claim 13 , wherein the cell is a cell of a mammalian cell line. 
     
     
         16 . The method of  claim 13 , wherein the cell is obtained from a mammal suffering from or suspected of suffering from a chromosomal breakage disorder. 
     
     
         17 . The method of  claim 1 , wherein the matrix is agarose. 
     
     
         18 . A method for mapping chromosome fragile sites in a eukaryotic cell, the method comprising:
 (a) contacting a plurality of eukaryotic cells with an inhibitor of DNA replication;   (b) embedding the cells in a matrix;   (c) within the matrix, contacting chromosomal DNA of the cells with labeling reagents for a time and under conditions sufficient to permit direct labeling of the DNA at single-stranded regions and/or at sites of double-stranded breaks;   (d) isolating labeled DNA from the matrix;   (e) detecting labeled DNA, wherein the detection of labeled DNA indicates the location of single-stranded regions and/or sites of double-stranded breaks in the chromosomal DNA of said cells, whereby one or more chromosome fragile sites are mapped.   
     
     
         19 . The method of  claim 17 , further comprising the step of removing the inhibitor of DNA replication before embedding the cells in a matrix. 
     
     
         20 . The method of  claim 18 , wherein the labeling reagents comprise random primers and biotinylated dUTP. 
     
     
         21 . The method of  claim 18 , further comprising, after labeling step (c), fragmenting the chromosomal DNA. 
     
     
         22 . The method of  claim 21 , further comprising the step, after the fragmenting step, of contacting the fragments with streptavidin bound to a solid support, whereby labeled DNA fragments are isolated. 
     
     
         23 . The method of  claim 18 , wherein the detecting step (e) comprises contacting isolated, labeled DNA with a microarray, or subjecting isolated, labeled DNA to a high throughput DNA sequencing regimen. 
     
     
         24 . A kit for mapping chromosome fragile sites in the chromosomal DNA of a eukaryotic cell comprising:
 a matrix suitable for embedding eukaryotic cells; and   reagents for direct labeling of the DNA at single-stranded regions and/or at sites of double-stranded breaks.   
     
     
         25 . The kit of  claim 24 , further comprising control cells and/or control DNA. 
     
     
         26 . The kit of  claim 24 , further comprising at least one microarray.